Genomic DNA is continually bombarded with a variety of insults, resulting in damage that must be repaired. By necessity, cells have evolved mechanisms to detect and repair broken strands of DNA, thereby preventing loss of important genetic information. Double-stranded DNA breaks (DSBs) are a type of damage that lead to particularly disastrous effects if not corrected. Homologous recombination (HR) is a highly conserved pathway that cells can use to repair DSBs. The consequences of disrupting HR are devastating. For example, mutations in the Rad51 recombinase are embryonic lethal in mice, and mutations in human Rad51 are linked to breast cancers. In addition, defects in BRCA2 account for at least 5% of all breast cancers and also confer a genetic predisposition to ovarian cancer. BRCA2 is thought to help regulate HR, and loss of this regulation may be the reason why this gene is linked to hereditary cancers. When a DSB occurs, the DNA ends are processed to generate 3' single-strand DNA (ssDNA) overhangs. The ssDNA ends then pair with homologous sequence elsewhere in the genome, and the missing DNA sequence is replaced using the homologous DNA as a template for replication. Finally, the replicated intermediate is resolved, regenerating the continuity of the broken DNA. While seemingly simple, HR requires the coordinated action of a complex repertoire of proteins, which are responsible for sensing damage, recruiting essential factors, and processing and repairing the damaged DNA. Our overall goals are to understand how these proteins sense and respond to damaged DNA. To help address this problem we have developed unique technologies that allow us to directly visualize hundreds of individual DNA molecules at the single molecule level using optical microscopy. Here we will try to determine how proteins process the ends of DNA at the early stages of HR, determine how these enzymes act on crowded substrates that reflect physiological settings, and determine how end processing is coupled to assembly of other protein complexes that are necessary to complete the repair pathway. We will reveal the spatial and temporal progression of these events by directly watching individual biochemical reactions in real time, and part of the significance of this project lies in the depth of the answers we strive to obtain.